Line volume calibration systems and methods

ABSTRACT

Provided are systems and methods for line volume calibration, and measurement of fluid samples delivered to an interrogation point. In various embodiments, a known fluid volume comprising a sample line fluid and a secondary fluid is delivered to a fluid boundary sensor. The fluid boundary sensor assists in determining the position of the boundaries between the various fluids, and the positions of these boundaries are used to determine the sample line fluid volume.

RELATED APPLICATIONS

The present application is a continuation of now-allowed U.S. patentapplication Ser. No. 16/892,502, “Line Volume Calibration Systems AndMethods” (filed Jun. 4, 2020); which application claims priority to andthe benefit of U.S. patent application No. 62/858,507, “Line VolumeCalibration Systems And Methods” (filed Jun. 7, 2019). The foregoingapplications are incorporated herein by reference in their entiretiesfor any and all purposes.

TECHNICAL FIELD

The present disclosure generally relates to line volume measurements forfluidic samples.

BACKGROUND

When analyzing very small sample volumes, e.g., on the microliter scale,fluidic analysis instruments must be very precise when measuring thesample volume and collecting data. Flow cytometry, for example, cananalyze single cells suspended in fluid, as the fluid passes by a highlyfocused laser beam, and obtains information about the cells based on thescattered light. In such experiments, the precise volume (i.e., linevolume) of the fluid sample must be known to accurately correlate thetiming of the scattered light (e.g., forward scattered light, sidescattered light) from cells and also fluorescence from fluorescentlabels associated with cells of the fluid sample. Likewise, the precisetiming of when the sample enters and exits the interrogation region isessential for determining start/stop times for data collection andanalysis. Thus, having an accurate determination of the fluid linevolume between the sample origination point and the interrogation pointis critical to system performance.

Current methods to address these challenges include beginning ananalysis of the sample fluid late and ending the analysis earlier thanthe actual beginning and end of the sample. Cutting off analysis of theleading and trailing ends as the sample fluid passes the interrogationpoint ensures that it is sample fluid that is being analyzed, not air orother non-sample fluid in the line.

A shortcoming of this approach, however, is that a portion the samplefluid is wasted and is not analyzed. This can be particularlyproblematic during experiments attempting to detect and analyze rareevents (e.g., cells in a sample fluid expressing a rare protein), asrare event analytes that are present in only dilute quantities can bemissed if some of the sample fluid that contains such analytes isdiscarded or not analyzed. Further, some events of interest may not endup being analyzed because of their location at the beginning or end ofthe fluid sample.

Careful sample line manufacturing methods are intended to provideconsistent and reliable sample line volumes, as such carefulmanufacturing methods attempt to ensure that sample line volumes fallwithin a certain tolerance or range. This approach, however, oftenresults in a higher cost of manufacturing and testing of sample lines.In addition, during analysis, sample arrival times must be calculatedwith larger margins of error to allow for variations in the line volume.This in turn results in waste of potentially valuable sample, andportions of both ends of the analysis period must be discarded (and withsuch discard, the potential loss of valuable information that could becritical for both research purposes as well as clinical purposesassociated with diagnosing a patient).

Accordingly, there is a long-felt need in the art for improved methodsand systems of line volume calibrations and sample measurements.

SUMMARY

In meeting the described challenges, the present disclosure firstprovides systems and methods for measuring, estimating, and/ordetermining a volume of sample. A method can comprise: disposing acalibration volume of a first fluid into the sample line such that thecalibration volume completely fills the sample line, the completelyfilled sample line defining a volume SL therein; communicating into asample zone the calibration volume of the first fluid and also adisplacement volume of a second fluid, the calibration volume of thefirst fluid and the displacement volume of the second fluid defining atotal volume; communicating the total volume from the sample zone to asensor configured to identify a boundary between the first fluid and thesecond fluid; and from the boundary, estimating the volume SL of thesample line.

The present disclosure also provides systems and methods, comprising: asample line enclosing a volume SL therein; a sample zone configured toreceive a first fluid from the sample line; a fluid delivery trainconfigured to (a) deliver a volume of the first fluid into the sampleline, (b) deliver a calibration volume CV of the first fluid thatcompletely fills the sample line from the sample line into the samplezone, and (c) deliver a displacement volume D of a second fluid into thesample zone such that the calibration volume CV of the first fluid andthe displacement volume D of the second fluid define a total volume TV;a sensor region configured to receive the first fluid and the secondfluid from the sample zone and to detect a boundary between the firstfluid and the second fluid; a flow diverter train configured to (a)place the sample line into fluid communication with the sample zone, (b)place the sample zone into fluid communication with the sensor, or both(a) and (b); and optionally, a processor configured to determine avolume enclosed by the first sample line based on at least a differencebetween the volume D of the displacement volume of the second fluid andthe total volume TV.

Additionally provided are systems and methods for estimating a volume ofa sample fluid, comprising: delivering an amount of a first fluid to aconduit; delivering an amount of a second fluid into the conduit so asto displace the first fluid within the conduit; estimating a totalvolume of the first fluid and the second fluid in the conduit;delivering the first fluid and the second fluid from the conduit to asensor capable of determining a boundary between the first fluid and thesecond fluid; determining a volume of the second fluid in the totalvolume; and estimating a volume of the sample fluid based on at leastthe volume of the first fluid and the estimated total volume of thesample fluid and the first fluid.

Also provided are systems for automatically determining the volume of asample, the system comprising: a sensor region; a fluid delivery trainconfigured to separately deliver a volume of a second fluid and a volumeof a first fluid into the sensor region, the sensor region beingconfigured to measure a signal through the sensor region, the signaldiffering based on the presence of the first fluid in the sensor regionor the presence of the second fluid in the sensor region; and aprocessor configured to determine a volume of the sample based on thesignal measured when the first fluid and the second fluid arecommunicated through the sensor region.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the disclosed subject matter, there are shown inthe drawings exemplary embodiments of the disclosed subject matter;however, the disclosed subject matter is not limited to the specificmethods, compositions, and devices disclosed. In addition, the drawingsare not necessarily drawn to scale. In the drawings:

FIGS. 1A-1I provide a depiction of an exemplary system and methodaccording to the present disclosure;

FIG. 2 provides a depiction of an exemplary embodiment according to thepresent disclosure; and

FIG. 3 provides a flowchart of an embodiment of the disclosedtechnology.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure can be understood more readily by reference tothe following detailed description taken in connection with theaccompanying figures and examples, which form a part of this disclosure.It is to be understood that this disclosure is not limited to thespecific devices, methods, applications, conditions or parametersdescribed and/or shown herein, and that the terminology used herein isfor the purpose of describing particular embodiments by way of exampleonly and is not intended to be limiting of the claimed subject matter.

Also, as used in the specification including the appended claims, thesingular forms “a,” “an,” and “the” include the plural, and reference toa particular numerical value includes at least that particular value,unless the context clearly dictates otherwise. The term “plurality”, asused herein, means more than one. When a range of values is expressed,another embodiment includes from the one particular value and/or to theother particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another embodiment. All ranges areinclusive and combinable. It is to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting.

It is to be appreciated that certain features of the disclosed subjectmatter which are, for clarity, described herein in the context ofseparate embodiments, can also be provided in combination in a singleembodiment. Conversely, various features of the disclosed subject matterthat are, for brevity, described in the context of a single embodiment,can also be provided separately or in any subcombination. Further, anyreference to values stated in ranges includes each and every valuewithin that range. Any documents cited herein are incorporated herein byreference in their entireties for any and all purposes.

In one aspect, the present disclosure provides systems and methods formeasuring, estimating, and determining volumes of samples. Variousembodiments relate to instruments using a fluidic system to deliver asample for analysis to an interrogation point. In such systems, anaccurate determination of the fluid line volume between the sampleorigination point and the interrogation point is critical to systemperformance. Without accurate measurements, the arrival time of thesample at the interrogation point is indeterminate, and can result inpotential data loss or corruption, as discussed herein.

Accordingly, the present invention provides many unique advantages overtraditional systems and methods. With respect to manufacturing, forexample, variations in manufacturing are compensated for by determiningthe line volume after the system is complete. By having a more precisemeasurement of the line volume, a greater percentage of the sample canbe used for analysis. This is particularly important in cases wherethere is a small amount of sample for use. In addition, if components ofthe system are changed at a customer site, the line volume can be easilydetermined again to allow for any variation that may occur.

In various embodiments, the line volume determinations and samplemeasurement systems and methods can be installed in implemented in oneor more existing systems. That is, the disclosed embodiments are notlimited to the depicted illustrations and expels, and can be applied tocurrent analysis systems, e.g., flow cytometers, and improve analysesand data collection.

FIGS. 1A-1I illustrate an example system and method for determining avolume enclosed within a sample line. In embodiments, as discussedherein, the sample line can contain a sample fluid for analysis in oneor more instruments.

Figure Elements Legend

For convenience, the following is a list of elements used in connectionwith FIGS. 1A-1I:

-   102: Fluid (e.g., focus fluid) tank-   104: First fluid (e.g., focus fluid)-   106: Pump (configured to communicate fluid 104 from fluid tank 102)-   108: Flow diverter (e.g., valve)-   110: Fluid boundary sensor (configured to detect a signal related to    a boundary between fluids, e.g., a boundary between focus fluid and    air)-   112: Pump (configured to draw fluid into fluid boundary sensor 110)-   114: Pump (configured to draw fluid into sample zone 116, can also    be configured to exert fluid out of sample zone 116)-   116: Sample zone—a sample zone can be, e.g., within a flow cytometer    analyzer (with acoustic and/or hydrodynamic based focusing of cells    and other particles), imager, flow cytometer cell and other particle    sorter/separator, and the like.-   116 a: Volume of fluid that fills entirety of sample line 118 out to    end of sample line head 122-   116 b: Second fluid (e.g., air bubble) communicated into sample zone    116 following communication of sample line volume 116 a into sample    zone 116-   118: Sample line (shown with dashed line for ease of reference)-   120: Overflow vessel (configured to receive fluid exiting sample    line head 122 of sample line 118)-   122: Sample line head (shown with dashed line for ease of reference)-   124: Pump (e.g., configured to draw fluid from overflow vessel and    communicate the fluid to tank 126)-   126: Tank

FIG. 1A shows an initial state of a system that includes a sample linehaving a sample line volume to be determined. A first fluid (e.g., focusfluid) 104 can be contained in fluid tank (e.g., a focus fluid tank)102. As shown, portions of focus fluid can be drawn from fluid tank 102towards sample line 118. In various embodiments, the sample line and theother conduits through which fluid flows can be tubing comprising one ormore of a variety of materials.

As illustrated in FIG. 1B, pump 106 draws first fluid 104 out of fluidtank 102 and towards a flow diverter 108 (which can be, e.g., a valve orrotary valve), and sample line 118. (For ease of reference, the presenceof first fluid 104 is represented by a heavy dark line.)

Flow diverter 108 can be in a state whereby fluid is communicatedthrough one or more fluid lines (not labeled) from tank 102 to sampleline 118; as one example, flow diverter 108 can be in a state wheresample line 118 is in fluid communication with tank 102 but sample line118 is not in fluid communication with sample zone 116. It should beunderstood that flow diverter 108 can be a single unit (e.g., a singlevalve capable of multiple states). In some embodiments, flow diverter108 can comprise multiple units (e.g., multiple valves).

Sample line 118 contains the total volume of fluid to be determined. Insome embodiments, the focus fluid volume acts as a calibration volume.Sample line 118 can further include a sample line head 122 that in someembodiments is contained in (or empties into) overflow vessel 120configured to receive excess fluid existing the end of the sample line118. It will be appreciated that total volume of the sample line 118includes the volume contained within the filled sample line head 122,which sample head is also the end of the sample line 118.

When drawing focus fluid into the sample line 118, as seen in FIG. 1C,excess focus fluid is drawn and deposited into overflow vessel 120. Inembodiments, this can occur because the total volume of the sample lineis initially unknown, so excess fluid is pumped through to ensure thatthe sample line 118 is completely filled. In some embodiments, theoperational precision of pump 106 can vary, thus drawing excess focusfluid 104 into the sample line. It is considered especially suitable tooperate such that sufficient fluid is communicated to sample line 118such that sample line 118, including the sample head 122, is fullyfilled. Excess fluid will exit sample line head 122 into overflow vessel120, which can be in fluid communication with the end of the sampleline. In some embodiments, a fluid delivery train can be utilized toremove fluid from the overflow vessel 120 while fluid is retained withinthe sample line 118 and sample line head 122.

In some embodiments, the amount of excess fluid in the sample line head122 and overflow vessel 120 is minimized. One or more sensors in thesample line head 122 and overflow vessel 120 can provide feedback to oneor more components of the depicted system, including but not limited tothe pump 106, to indicate when the sample line head is filled and/orwhen excess fluid has been realized in the overflow vessel 120, suchthat the amount of wasted focus fluid is minimized. In this way, asystem can operate in an automated fashion such that, e.g., when excessfluid exits sample line head 122 and is detected, the system ceasessupplying further fluid to sample line 118 and clears excess fluid thathas exited sample line head 122 and has accumulated in vessel 120.

As shown, FIG. 1D illustrates that any excess fluid in the overheadvessel can be communicated to waste tank 126 by fluid delivery train 124(e.g., a pump). Train 124 is thus configured to draw fluid from theoverflow vessel 120 and communicate the fluid to a waste tank 126 orother waste area. It will be appreciated that the train 124 can beconfigured so as not to remove any focus fluid from sample line 118.Thus, sample line 118 remains completely filled with fluid 104 all theway through the end of the sample line at sample line head 122; thevolume of fluid filling sample line 118 to the end of the line is shownby 116 a.

After the sample line 118 has been completely filled, pump 114 canoperate to communicate the focus fluid contained in the sample line intoa sample zone 116, which can be a sample loop. (It should be understoodthat although 114 is referred to as a pump for convenience, 114 caninclude one or more pumps, valves, and the like.)

FIG. 1E illustrates sample line volume fluid 116 a after fluid 116 a hasbeen communicated to sample zone 116. In addition to the fluid 116 afrom the sample line, pump 114 can also draw a second fluid, e.g., anair bubble, “behind” sample line fluid volume 116 a. The second fluid,is shown by bubble 116 b within sample zone 116. Thus, the total volumedrawn by pump 114 into the sample zone 116 is the sample line fluidvolume 116 a plus the volume of fluid bubble 116 b.

In some embodiments, pump 114 can be a pump (e.g., a syringe pump orother precise modality) that is configurable to draw up a predeterminedand/or user-designated amount of fluid, e.g., 450 μl. In other words,pump 114 can draw a known amount of fluid (comprising both the sampleline fluid volume 116 a and air bubble 116 b) into sample zone 116.Thus, the amount 116 c of fluid drawn into the sample zone 116 is known,as this amount 116 c is the amount of fluid that pump 114 has drawn intosample zone 116.

FIG. 1F illustrates that pump 112 draws the fluid within the sample zone116 (comprising the sample line fluid volume 116 a and air bubble 116 b)through flow diverter 108 (e.g., a rotary valve), towards fluid boundarysensor 110. (As shown in FIG. 1F, bubble 116 b is about to enter sensor110, with sample volume 116 a following bubble 116 b.) The fluidboundary sensor is configured to detect a signal related to a boundarybetween fluids. In some embodiments, the fluids are immiscible fluids.That is, the sensor, which can detect signals, such as optical signals,electric signals, acoustic signals, or any combination thereof, candetermine a first signal related to a first fluid (e.g., air, bubble 116b, etc.) or calibration fluid, then when the first fluid passes through,the signal obtained by the boundary sensor changes, as the propertiesand qualities of the focus fluid are distinct from the initialcalibration fluid's measurement.

In some embodiments, the fluid boundary sensor is a bubble sensor. Insome embodiments, the fluidic pathway between sample zone 116 and fluidboundary sensor 110 is pre-filled with a prefill fluid, e.g., a fluidthat is the same as first fluid 104. The prefill fluid can be a fluidthat is immiscible with air bubble 116 b.

When pump 112 has drawn bubble 116 b completely into the fluid boundarysensor 110, as illustrated in FIG. 1G, sensor 110 can determine thestart and end points of each of the bubble 116 b and the sample linefluid volume 116 a. (As shown in FIG. 1G, bubble 116 b has enteredsensor 110, and sample line volume 116 a is about to enter sensor 110;the sensor is configured to detect the “beginning” and “end” of bubble116 b.)

As pump 112 continues to draw sample line fluid volume 116 a into thefluid boundary sensor 110 (FIG. 1H), the start and end points of thesample line fluid volume 116 a, as well as any additional bubbles, ifany, that may be contained within the sample line fluid volume. Invarious embodiments, the presence of additional bubbles once the sampleline fluid is first identified can indicate an air leak and/or problemwith one or more components of the system (e.g., a hole in one or moretubing) that allows air bubbles to be introduced.

Accordingly, the use of the fluid boundary sensor provides the start andend times of each of the various fluids initially contained in thesample zone. From the fluid boundary information, as well as the knownvolume (116 c, shown in FIG. 1E) drawn into sample zone 116, the userand/or system can determine the volume of bubble 116 b, and from thatthe sample line fluid volume 116 a by subtracting the volume of bubble116 b from the total drawn volume 116 c, i.e., 116 c=116 a+116 b.

In various embodiments, the readings from the fluid boundary sensor canbe analyzed at a computing device comprising a processor thatdetermines, from the boundary readings, the volume of bubble 116 b andsample line fluid volume 116 a in the manner provided above. Forexample, certain aspects can be executed by a machine, such as acomputer, through program code execution. Program code (i.e.,instructions) embodied in tangible storage media or memory mediaimplemented as storage devices, such as magnetic or optical media,volatile or non-volatile media, may be included. One or more programsthat may implement or utilize the processes described in connection withthe disclosure, e.g., through the use of an application programminginterface (API), reusable controls, or the like, and can automate one ormore operations disclosed herein.

FIG. 1I illustrates the pump 112 drawing all of sample line fluid volume116 a through the fluid boundary sensor 110. After passing through theboundary sensor 110, the sample line fluid volume should be known, andthe sample fluid can be delivered to one or more conduits for use.Sample line volume 116 a can be drawn through sensor 110 (after bubble116 b has been drawn through sensor 110) so that sensor 110 can detectthe presence of any other bubbles (or other occlusions) in sample linefluid 116 a. Without being bound to any particular theory, the presenceof one or more bubbles in sample line fluid 116 a can be indicative of aleak (e.g., an air leak) somewhere in the system, and the identificationof bubbles in sample line fluid 116 a can thus be used as a systemdiagnostic that can in turn inform the user regarding the presence (orabsence) of leaks in the system.

It will be appreciated that the disclosed systems and methods are notlimited to the specific depictions illustrated in FIGS. 1A-1I. Inaddition, the drawings are not necessarily drawn to scale, and the textlabels on the various parts of the drawings are illustrative only andare not limiting of the disclosed technology.

In various embodiments, the pumps described herein can be any of avariety of pumps, including but not limited to syringe pumps anddiaphragm, for drawing up fluid to its intended location. In addition,any of a variety pumps can be used to load a user-specified orpredetermined amount of fluid can be implemented. For each pumpreferenced in the Figures, one or more pumps can be utilized inaccordance with embodiments described herein. It will be appreciatedthat the depicted examples are not limiting to the embodiments providedin this disclosure.

FIG. 2 provides a depiction of another exemplary system 200 inaccordance with the present disclosure.

In the depicted embodiment, an auto sampler 206 and an instrument 220.The instrument can be a flow cytometer analyzer, e.g., an Attune™ NXTflow cytometer analyzer (Thermo Fisher Scientific Inc.), other flowcytometer analyzer, flow cytometer sorter/separator, or other fluidicinstrument. The auto sampler 206 and instrument 220 are utilized inaccordance with the disclosed systems and methods. As shown, a samplefluid, e.g., focus fluid, stored in a storage volume 204 (e.g., a bottleor other container) is drawn up through line 208 by a pump 210 (whichpump can be, e.g., a syringe pump) through a line (212/212 a/212 b) to asample line (214, 214 a, 214 b) that spans the auto sampler 206 andinstrument 220 (e.g., a cytometer). As shown, the auto sampler 206 andinstrument 220 can be located at a distance from one another, whichdistance can be spanned by line 212/212 a. Pump 218 (which can be, e.g.,a diaphragm pump) can encourage the sample fluid into sample line214/214 a/214 b. Pump 218 pump can comprise one or more check valves soas to ensure fluid flow in only a single direction. Pump 218 can also beconfigured so as to completely fill the sample line. A container 230 canreceive sample fluid from line 214. Line 216 (if present) can connectcontainer 230 to pump 218. Instrument 220 can include a sample injectionport and/or one or more sample holders; a sample holder can accommodatetubes or other sample containers.

Excess focus fluid at the end of sample line 214 can be collected anddelivered into a waste container 202. As an example, a diaphragm pumpcan draw excess fluid from the head of sample line 214 to wastecontainer 202. At this point, the exact volume of the sample line may ormay not be known.

Valve 224 can be configured so as to admit sample fluid in line 214/214a/214 b to sample loop 226, without also admitting the sample fluid toline 212 b or line 228. The sample fluid can be encouraged into sampleloop 226 by a pump, which pump can be a pump that delivers a knownamount of fluid to the sample loop. The known amount of fluid can be,for example, all of the sample fluid that resides in line 214/214 a/214b plus an additional amount of another fluid, e.g., air or other gas, orother fluid. The other fluid can be a fluid that is immiscible with thesample fluid.

As an example, after the sample line 214/214 a/214 b is filled, a pumpconnected to valve 224 (which can place sample loop 226 and sample line214/214 a/214 b into fluid communication with one another), draws thesample fluid from sample line 214/214 a/214 b up into sample loop 226along with an air bubble; the total volume of fluid (i.e., the sampleline fluid volume plus the air bubble) is known.

The rotary valve can be actuated to place the sample loop and bubblesensor into fluid communication with one another), and a pump can thenbe used to draw the air bubble and sample fluid from the sample loopthrough the bubble sensor. The measurements from the bubble sensoridentifying the boundary positions of the air bubble and sample fluid,as well as the known amount drawn up into the sample loop, can be usedto determine to determine a volume of the air bubble, and a volume ofthe sample fluid. The volume of the sample fluid can then correspond tothe volume in the sample line, when the sample line is completelyfilled.

As an example, a pump can be configured to draw into the sample loop 450μl of fluid, for example. (The total amount drawn into the sample loop,i.e., the sample fluid plus air, will be greater than the total amountcontained in the sample line so as to ensure that an air bubble ispresent within the sample loop.) In some scenarios, an amount offocusing fluid and air can be estimated (e.g., 370 μl fluid, 80 μl air)based on one or more known factors about the system, and in others, theamount of each fluid is unknown.

A valve (e.g., a rotary valve) is then modulated to direct the fluidfrom the sample loop 226 towards bubble sensor 222. As the air bubbleand fluid pass through bubble sensor 222, the start and end positions ofeach of the bubble and the focus fluid are recorded. From thatinformation, as well as the known amount of air and focus fluidcontained in the sample loop, the size of the bubble can be determined.For example, if, based on the bubble sensor readings it was determinedthat the air bubble volume is 83 μl, then the actual sample line fluidvolume would be 367 μl (i.e., 450 μl— 83 μl=367 μl).

It should be understood that although the numbers in the present exampleare in the range of hundreds of microliters, the disclosed systems andmethods are not limited to the microliter range. For example, one canapply the disclosed technology concepts to determine hundreds ofmilliliters or more, based on the system and the volume size to bedetermined.

Further, the size of the bubble is not critical, and is ultimatelyconnected to the type of bubble sensor (or other fluid boundary sensor),its sensitivity, and intended purpose. For example, use of an ultrasonicbubble sensor, e.g., for flow cytometry, may result in an attempt toensure that the bubble greater than 10 ul. However smaller bubbles canbe used. A user may wish to have a bubble larger than a minimum sizebased on the sensor, and to be able to ensure that the whole bubble isdetected.

Valve 224 can then be modulated so as to admit the sample fluid (andother fluid) from sample loop 226 to line 228 (but not to lines 212 b or212 b). The sample fluid is then communicated to sensor 222 (which canbe, e.g., a bubble sensor). As described elsewhere herein, the amount offluid in sample loop 226 is known, so the amount of fluid communicatedto sensor 222 from sample loop 226 is also known.

Sensor 222 then detects an interface between the sample fluid and theother fluid that is communicated to the sensor. By detecting theinterface, the system can then determine the volume of fluid that wasoriginally contained in sample line 214/214 a/214 b. In this way, theuser can—as described elsewhere herein—perform experiments with lesswaste and greater throughput, as the user will know how much samplefluid is being delivered to the sample loop for each experimental runbeing performed in the sample loop.

In some embodiments, the disclosed systems and processes could beconducted infrequently, such as at installation or when the system haschanged (e.g., after maintenance or service, after replacing one or moresystem components). Additionally, and/or alternatively, the disclosedsystems and methods can be utilized as a periodic maintenance activity(e.g., every few months or on another recurring schedule).

FIG. 3 provides an exemplary flowchart of method 300 according to thedisclosed technology. As shown, method 300 can include step 302 offilling a sample line (completely) with a first fluid. Such a fluid canbe, e.g., buffer, sheath fluid (e.g., for acoustic and/or hydrodynamicfocusing).

This can be accomplished by, e.g., overfilling the sample line, stoppingthe flow of first fluid to the sample line, and collecting any overflowfluid from the sample line, leaving behind the sample line completelyfilled. One can then (step 304) introduce to a holding region a totalvolume (TV) made up of the sample fluid from the completely filledsample line and a volume of a second fluid to a holding location. Theholding location can be, e.g., a sample loop, a container, or otherlocation. The TV of the first fluid and the second fluid can beintroduced to a sensor region (step 306), e.g., a tube, a vessel, or thelike. The sensor region can include a sensor—such as a bubblesensor—that can detect a boundary between two fluids, e.g., air andwater. A bubble sensor can operate by ultrasound, by capacitance, or byother modes known to those in the art. The total volume (TV) of thefirst fluid and second fluid can be communicated through the sensorregion, e.g., at a known flowrate. The sensor can detect (step 308) thepresence of a boundary between fluids (e.g., between the second fluidand the sample or first fluid). From the detection of a boundary (step310), one can (manually or in an automated fashion) determine the volumeof one (or both) of the first fluid and the second fluid in the TV.

As an example, if TV is known to be 500 μL, the TV is passed through thesensor region at 100 μL per minute, and a bubble sensor detects thefront boundary of the TV (i.e., where the second fluid begins) at t=0sand a boundary between the first and second fluids at t=1 minute afterthe front boundary, then one can determine that the TV includes 100 μLof the second fluid and 400 μL of the first fluid. This in turnestablishes that the sample line defined a volume of 400 μL, the volumeof the first fluid in the TV. One can (step 312) perform furtheranalysis (e.g., flow cytometry), as one would then know the volume ofthe fluid in the (fully filled) sample line. In this way, one knows theprecise amount of fluid being (i.e., 400 μL, in the foregoing example)delivered from the sample line to the sample zone for each analysisbeing performed in the sample zone.

Exemplary Aspects

The following Aspects are illustrative only and should not be understoodas limiting the scope of the present specification or the scope of theappended claims.

Aspect 1. A method comprising: disposing a calibration volume of a firstfluid into the sample line such that the calibration volume completelyfills the sample line, the filled sample line defining a volume SLtherein; communicating into a sample zone the calibration volume of thefirst fluid and also a displacement volume D of a second fluid, thecalibration volume of the first fluid and the displacement volume D ofthe second fluid defining a total volume; communicating the total volumefrom the sample zone to a sensor configured to identify a boundarybetween the first fluid and the second fluid; and from the boundary,estimating the volume SL of the sample line.

Fluidic lines (e.g., the sample line) can be formed of flexible or rigidmaterials. Flexible tubing is considered suitable, but is not arequirement.

It should be understood that one or more of the steps of the disclosedmethods can be performed in an automated fashion. As an example, thestep of communicating the first and second fluids into the sample zonecan be performed in an automated fashion.

As shown in FIGS. 1A-1I, fluid can be drawn into a sample line, and thevalve that places the sample line into fluid communication with thesample line can be closed such that the sample line (118, in FIG. 1C) iscompletely filled. The sample line's end can be immersed in a catchmentcontainer (112, in FIG. 1C), which is then emptied so as to leave behindthe sample line in a completely filled state.

Aspect 2. The method of Aspect 1, wherein the sensor detects a change inan electrical signal, a change in an acoustic signal, a change in anoptical signal, or any combination thereof. As an example, a sensor candetect a change in optical signal based the illumination received when aliquid is disposed within the sensor as compared to the illuminationreceived with air is disposed within the sensor. A sensor can also beconfigured to detect a change (e.g., a change in conductance, a changein optical signal, a change in acoustic signal) related to a boundarybetween a first fluid (e.g., air) and a second fluid (e.g. buffer). Thefirst fluid and the second fluid can be immiscible with each other.

Aspect 3. Any one of Aspects 1 or 2, further comprising communicatinginto the sample line a volume of the first fluid that exceeds a volumethat completely fills the sample line and removing first fluid thatexceeds the volume that fills the sample line so as to leave the sampleline completely filled. This can be performed in a manual or anautomated fashion.

Aspect 4. Any one of Aspects 1-2, further comprising communicatingthrough the sample line a volume of the first fluid that exceeds avolume that fills the sample line and then removing first fluid thatexceeds the volume that fills the sample line so as to leave the sampleline filled to the end of the sample line. This is shown in FIG. 1D andFIG. 1E, in which excess fluid exits sample line 118 and at leastpartially fills vessel 120 (FIG. 1D). That excess fluid is then removedfrom vessel 120 (FIG. 1E), leaving sample line 118 filled to the end.

Aspect 5. Any one of Aspects 1-4, wherein the first fluid and the secondfluid are immiscible. As an example, the first fluid can be a buffer,growth medium, sheath fluid (also termed a focus fluid in someinstances), or other fluid, and the second fluid can be air.

Aspect 6. Any one of Aspects 1-5, wherein the method is performed in anautomated fashion.

Aspect 7. Any one of Aspects 1-6, wherein the second fluid comprisesair.

Aspect 8. Any one of Aspects 1-7, further comprising operating thesample zone so as to analyze one or more fluid samples, each of the oneor more samples having a sample volume of the volume SL. A sample zonecan include, e.g., a particle concentration train (hydrodynamicconcentration, acoustic concentration, or both). A sample zone caninclude one or more sensors (e.g., a illumination emitter and detector)configured to interrogate one or more analytes disposed in a sample thatenters the sample zone.

As described elsewhere herein, in existing approaches, sample arrivaltimes are calculated with comparatively large margins of error to allowfor variations in the line volume. This in turn results in waste ofpotentially valuable sample, and portions of both ends of the analysisperiod must be discarded (and with such discard, the potential loss ofvaluable information that could be critical for both research purposesas well as clinical purposes associated with diagnosing a patient).Through application of the disclosed technology, a user can determine,with precision, the volume of a sample line and thus the volume of fluid(e.g., buffer, sheath fluid) that is in turn delivered to the sampleloop or zone, which sample loop or zone can include one or more analysisor processing modules, such as a flow cytometer, cell, sorter, and thelike.

By knowing the amount of fluid being delivered to the sample zone, auser can thus operate the sample zone more efficiently, without havingto discard significant portions of the “ends” of a sample in a givenanalysis period. For example, a user knowing that precisely 30 μL ofsample is delivered to the sample zone for each experimental run can inturn configure the sample zone to process the middle 29.5 μL of each 30μL portion of fluid delivered to the sample zone. If, on the other hand,a user (e.g., a user who uses existing approaches) knows only that thesample line contains from 25 to 35 μL of sample, the user may be forcedto conservatively configure the sample zone, e.g., to process only themiddle 20 μL of sample, a significant reduction in volume from thevolume that could be reliably processed using the disclosed technology.

The advantages of the disclosed technology are thus apparent, as knowingthe volume of the sample line allows the user to analyze a greaterproportion of a given sample, as the user need not build in a margin oferror and discard as much of the sample. A user can also increasethroughput, as a user can use less sample per experimental run, which inturn increases the number of experimental runs that can be performed pertime. Further, because one can use less sample per experimental run, onemay be able to obtain samples (blood, etc.) more easily, as less fluidwill be needed per sample. Thus, the disclosed technology also includesconfiguring (whether manually or in an automated way) the operation ofthe sample zone based on the determined volume of the sample line. Theconfiguring can include adjusting the sample zone to operate using lesssample, to operate with less discarding of sample (e.g., the sample“ends” of a given experimental run), or other adjustment. By using lesssample per experimental run, a user can also perform experiments withthe use of less reagents.

Aspect 9. A system, comprising: a sample line enclosing a volume SLtherein; a sample zone configured to receive a first fluid from thesample line; a fluid delivery train configured to (a) deliver a volumeof the first fluid into the sample line, (b) deliver a calibrationvolume (CV) of the first fluid that completely fills the sample linefrom the sample line into the sample zone, and (c) deliver adisplacement volume D of a second fluid into the sample zone such thatthe calibration volume CV of the first fluid and the displacement volumeD of the second fluid define a total volume TV; a sensor regionconfigured to receive the first fluid and the second fluid from thesample zone and to detect a boundary between the first fluid and thesecond fluid; a flow diverter configured to (a) place the sample lineinto fluid communication with the sample zone, (b) place the sample zoneinto fluid communication with the sensor, or both (a) and (b); andoptionally, a processor configured to determine a volume enclosed by thesample line based on at least a difference between the volume D of thedisplacement volume of the second fluid and the total volume TV.

A fluid delivery train can include one or more pumps, valves, flowdiverters, and the like. Pumps can be syringe pumps, gear pumps, and thelike. It should be understood that a pump can act to expel fluid, butcan also act to draw fluid in. As an example (and by reference to FIGS.1D, 1E, and 1F), pump 114 can act to draw sample line fluid 116 a intosample zone 116, along with bubble 116 b. Pump 114 can then act to expelfluid 116 a and bubble 116 b from sample zone and encourage fluid 116 aand bubble 116 b out of sample zone 116, through flow diverter 108, andtoward (and even into) sensor 110. Thus, pump 114 can be operated inforward and reverse modes.

By reference to FIGS. 1A-1I, pump 112 is optional. Likewise (and againby reference to FIGS. 1A-1I, the locations and operation of any of pumps106, 124, 114, and 112 can be optional, as one or more of the foregoingpumps may not be needed. Put another way, any of pumps 106, 124, 114,and 112 are optional, as the fluid delivery train of the disclosedsystems (and methods) can operate using one pump and can even operate ina gravity-based fashion. Through arrangement of valves, the disclosedsystems and methods can be operated by a single pump, though multiplepumps can be used.

Aspect 10. The system of Aspect 9, further comprising a vessel in fluidcommunication with an end of the sample line, the vessel beingconfigured to receive fluid communicated through the sample line.

Aspect 11. The system of Aspect 10, further wherein the fluid deliverytrain is further configured to remove fluid from the vessel while fluidis retained within the sample line such that the sample line iscompletely filled.

Aspect 12. The system of any one of Aspects 9-11, wherein the flowdiverter comprises a single valve.

Aspect 13. The system of Aspect 12, wherein the single valve ischaracterized as a rotary valve.

Aspect 14. The system of any one of Aspects 9-13, wherein the flowdiverter train comprises a plurality of valves. As an example, a flowdiverter train can include a valve that modulates flow between thesample line to the sample zone, and another valve that modulates flowbetween the sample zone and the sensor region.

Aspect 15. The system of any one of Aspects 9-14, wherein the sensorregion comprises a bubble sensor.

Aspect 16. The system of any one of Aspects 9-15, further comprising aninstrument (e.g., a flow cytometer) configured to analyze a sampledisposed in first fluid received by the instrument from the sample zone.

Aspect 17. The system of Aspect 16, wherein the system is configured tooperate the instrument based at least in part on a volume enclosed bythe sample line.

Aspect 18. The system of Aspect 17, further comprising an autosampler.

Aspect 19. The system of Aspect 18, wherein the autosampler is locatedat a distance from the instrument.

Aspect 20. The system of Aspect 19, wherein the sample line places theautosampler into fluid communication with the instrument. In this way, asystem can be configured to determine the volume of the sample line thatconnects the autosampler to the instrument. This can be accomplishedeach time an instrument and autosampler are connected, e.g., when aninstrument is connected to a new autosampler, such as when anautosampler is replaced. Likewise, this can be accomplished when aninstrument is replaced but the autosampler remains in place.

Aspect 21. The system of any one of Aspects 9-20, wherein the systemcomprises a region configured to contact a sample and the first fluid.

Aspect 22. The system of Aspect 21, wherein the sample comprises cells,cellular components, or both.

Aspect 23. The system of any one of Aspects 9-22, wherein the volumeenclosed by the sample line is in the range of from about 30 microlitersto about 2.5 milliliters, e.g., from about 50 microliters to about 2milliliters, from about 75 microliters to about 1.75 milliliters, fromabout 100 microliters to about 1.5 milliliters, from about 200microliters to about 1.5 milliliters, from about 250 microliters toabout 1.25 milliliters, from about 350 microliters to about 1.1milliliters, from about 450 microliters to about 950 microliters, fromabout 550 microliters to about 850 microliters, or even from about 650microliters to about 725 microliters.

Aspect 24. A method for estimating a volume of a first fluid, the methodcomprising: delivering an amount of a first fluid to a conduit;delivering an amount of a second fluid into the conduit so as todisplace the first fluid within the conduit; estimating a total volumeof the first fluid and the second fluid in the conduit; delivering thefirst fluid and the second fluid from the conduit to a sensor capable ofdetermining a boundary between the first fluid and the second fluid;determining a volume of the second fluid in the total volume; andestimating a volume of the first fluid based on at least the volume ofthe first fluid and the estimated total volume of the first fluid andthe second fluid.

Aspect 25. The method of Aspect 24, wherein the first fluid comprisesair.

Aspect 26. The method of any one of Aspects 21-22, further comprisingoperating an instrument based at least in part on the estimated volumeof the first fluid, the instrument optionally being configured for fluidcommunication with the conduit.

Aspect 27. The method of Aspect 23, wherein the instrument is a flowcytometer.

Aspect 28. The method of any one of Aspects 26-27, wherein the firstfluid is communicated to the instrument from a sample line.

Aspect 29. The method of Aspect 28, wherein the volume of the firstfluid delivered to the instrument is a volume enclosed by the sampleline.

Aspect 30. The method of any one of Aspects 28-29, wherein the sampleline is disposed so as to place an autosampler in fluid communicationwith an instrument.

Aspect 31. The method of Aspect 30, wherein the autosampler is locatedat a distance from the instrument.

Aspect 32. The method of any one of Aspects 21-31, wherein thedetermining the volume of the second fluid in the total volume is based,at least in part, on detecting the boundary between the first fluid andthe second fluid.

Aspect 33. A system for automatically determining the volume of asample, the system comprising: a sensor region; a fluid delivery trainconfigured to separately deliver a volume of a second fluid and a volumeof a first fluid into the sensor region, the sensor region beingconfigured to measure a signal through the sensor region, the signaldiffering based on the presence of the first fluid in the sensor regionor the presence of the second fluid in the sensor region; and aprocessor configured to determine a volume of the sample based on thesignal measured when the first fluid and the second fluid arecommunicated through the sensor region.

Aspect 34. The system of Aspect 33, wherein the first fluid comprisesair.

Aspect 35. The system of any one of Aspects 33-34, further comprising aninstrument and optionally a flow modulator, the system being configuredto place the instrument into fluid communication with the sensor region.

Aspect 36. The system of Aspect 35, wherein the instrument comprises aflow cytometer, the flow cytometer optionally being characterized as anacoustic flow cytometer.

Aspect 37. The system of any one of Aspects 35-36, further comprising asample line, the system being configured to place the sample line intofluid communication with the instrument.

Aspect 38. The system of Aspect 37, wherein the instrument is operablebased on an estimated volume of the sample line.

Aspect 39. The system of any one of Aspects 37-38, further comprising acollection train configured to collect excess fluid from the sample lineso as to leave the sample line completely filled.

Aspect 40. The system of any one of Aspects 37-38, further comprising asource of the first fluid, the system being configured to place thesource of first fluid into fluid communication with the sample line.

Aspect 41. The system of any one of Aspects 33-40, wherein the system isconfigured to contact the first fluid with a sample.

Aspect 42. The system of Aspect 41, wherein the sample comprises cells,cellular components, or both.

What is claimed:
 1. A method for estimating a volume of a first fluid, comprising: delivering an amount of the first fluid to a sample line; delivering an amount of a second fluid into the sample line so as to displace the first fluid within the sample line; estimating a total volume of the first fluid and the second fluid in the sample line; delivering the first fluid and the second fluid from the sample line to a sensor capable of determining a boundary between the first fluid and the second fluid; determining a volume of the second fluid in the total volume; and estimating a volume of the first fluid based on the boundary and the estimated total volume of the first fluid and the second fluid.
 2. The method of claim 1, further comprising operating an instrument based at least in part on the estimated volume of the first fluid, the instrument optionally being configured for fluid communication with the sample line.
 3. The method of claim 2, wherein the instrument is a flow cytometer.
 4. The method of claim 2, wherein the volume of the first fluid delivered to the instrument is a volume enclosed by the sample line. 